Measuring the binding affinity between materials, molecules, and cells is key to a broad spectrum of industries, including but not limited to, material development, semiconductor production, bioanalytical assays, biomedical diagnostics, and drug discovery. With the emergence of solid state array-based bioanalytical and genetic diagnostic instruments and related equipment, new methods for cost effective screening of a large number of reactions in a miniaturized solid state form have become increasingly desirable.
The favored approach to date is to monitor changes in optical properties, usually fluorescence, when a known, fluorescently labeled molecule interacts with a known molecular species at a specific address in a molecular array. These apparatuses and methods, however, often impose stereochemical constraints by the addition of reporter systems to the molecules used to interrogate the molecular array. Furthermore, these methods do not directly report the actual binding affinity. Thus, label free, direct interrogation of molecular binding affinities using a micromechanical reporter is of obvious utility. More sophisticated and robust methods of this interrogation are required to better analyze a range of different objects bound, adsorped, or otherwise attached to a variety of surfaces.
One method for the direct detection of whether a molecule or other object is bound to a surface, or an object on the surface, is the scanning probe microscope. One type of scanning probe microscope is the atomic force microscope (“AFM”). In the AFM, a sharp probe is situated at the end of a flexible cantilever and scanned over a sample surface. While scanning, the probe and cantilever are deflected by the net sum of the attractive and repulsive forces between a tip of the probe and the surface and/or objects deposited on the surface. The deflection of the cantilever is usually measured by the reflection of a focused laser beam from the back of the cantilever onto a split photodiode, constituting an “optical lever” or “beam deflection” mechanism. The change in deflection indicates the presence of an object on the surface. These previous methods utilized materials bound to the probe to indicate the resultant force interactions between the probe and the material bound on the surface. Other methods for the detection of cantilever deflection include interferometry and piezoelectric strain gauges.
The first AFMs recorded only the vertical displacements of the cantilever. More recent methods also involve recording the torsional force, resonating the tip and allowing only transient contact, or in some cases no contact at all, between the probe and the sample. Plots of the tip displacement of the probe, or resonance changes as it traverses a sample surface, are used to generate topographic images. Such images have revealed the three dimensional structure of a wide variety of sample types including material, chemical, and biological specimens. Some examples of the latter include DNA, proteins, chromatin, chromosomes, ion channels, and even living cells. These prior methods, however, are limited to those complexes that can be bound to the probe and then dragged across the various materials on a surface. A new method is needed that-is not limited in this manner.
In addition to its imaging capabilities, the AFM can make extremely fine force measurements. The AFM can directly sense and measure forces in the microNewton (10−6) to picoNewton (10−12) range. Thus, the AFM can apply forces to, and measure forces between, molecular pairs, and even single molecules. Moreover, the AFM can measure and apply a wide variety of other forces and phenomena, such as magnetic fields, thermal gradients and viscoelasticity. This ability can be exploited to map force fields on a sample surface, and reveal with high resolution the location and magnitude of these fields, as in, for example, localizing complexes of interest located on a specific surface. Furthermore, to make additional molecular force measurements, the AFM probe may be functionalized with a molecule of interest.
Previous methods of evaluating the mechanical force necessary to remove bound objects to a surface included combinatorial chemistry techniques like repetitively binding one or more objects to a surface followed by washing the objects away. In this manner a characterization of how well the objects bound or adsorped to the surface could be determined based on population averages. A need exists for a cost effective and practical improvement to this methodology that results in a characterization of the force required to move objects residing on a surface.
A need exists for a simple and efficient method of quickly assessing the affinity of a bound molecule, cell, or other object to a surface. This method should overcome the prior art limitations of having to drag the material across a surface while attached to a probe, or having to sequentially wash microtiter plates to determine the affinity based on overall population samples. This method should also allow for the determination of binding affinities between the object and other materials that are bound to the surface. Finally, a need exists for a method for eliminating the poorly bound objects on the surface of interest so that the more tightly bound objects can be harvested for further study.